The invention relates to an agent and process for treating body fluids in the immunological determination of neopterin using a specific antibody against neopterin and a detection system.
Since the discovery of neopterin in the urine of patients with malignant or viral diseases in 1979, neopterin has become established as a parameter for ascertaining the cellular immune status in a multitude of patients with many different kinds of diseases.
Neopterin belongs to the pteridine group of compounds, which are heterocyclic molecules with a wide distribution in the plant and animal kingdoms. While some biological functions of pteridines are well documented, the biological role of neopterin is thus far largely unknown.
Neopterin is produced by monocytes/macrophages following induction by interferon gamma; interferon gamma is in turn directly coupled to the activation of cell-mediated immunity, and is formed by activated T-lymphocytes, when these are stimulated.
Cellular immunity plays a decisive role in a series of diseases, such as viral infections, intracellular parasites, septicaemia, graft rejections, autoimmune disorders and neoplasias. Raised concentrations of neopterin in body fluids directly reflect the degree of activation of the cellular immune system. Thus, determination of the concentration of neopterin in body fluids is very suitable indeed for monitoring the cellular immune status.
The determination of neopterin in body fluids for the purpose of establishing the cellular immune status in the case of malignant tumours and/or viral diseases is described, for example, in EP 12 444. Methods which have hitherto been customary for determining neopterin in body fluids, preferably in serum and urine, are high pressure liquid chromatography (HPLC), (J. Chromatogr. 277, 61 (1982)) and radioimmunoassay (Chem. Biol. Pteridines, 815 ff. W. de Gruyter, Berlin-New York (1983)). Both methods have some disadvantages, however. Neopterin can only be determined by HPLC after a relatively elaborate processing procedure. Each analysis then lasts for about a further 10 to 15 minutes, so that the measurement of a relatively large number of samples, even with automation, is associated with much expenditure of time and effort. Radioimmunoassays may only be carried out in specially approved laboratories, and specific guidelines must be followed for the safe disposal of radioactively contaminated working solutions and wastes.
In a competitive immunoassay for determining neopterin, on which the invention is based, the inner surfaces of plastic vessels, wells of a microtiter plate, polystyrene latex, glass spheres, or magnetic particles are coated, as the solid phase, with a neopterin-specific antibody. For example, the neopterin-containing samples to be analyzed and a conjugate consisting of neopterin and a marker enzyme are pipetted into the wells of a microtiter plate. The neopterin in the sample and the conjugate compete for the limited binding sites on the solid phase. High concentrations of neopterin in the sample then lead to low binding of conjugate on the solid phase. This in turn leads, after a washing step to remove all unbound substances, to low color development in a subsequent substrate reaction which is specific for the marker enzyme employed.
Quantification of the neopterin concentration in the samples is effected using a calibration curve which is plotted with standards of precisely defined neopterin concentrations. The average concentration of neopterin in the serum of healthy persons is about 5 nmol/l ; values above 10 to 15 nmol/l are considered to be pathological. Because of the high sensitivity, of at least 1 nmol of neopterin/l, of the immunoassay which is required, the serum sample must be combined directly with the reaction partners, i.e. the serum must be employed in undiluted form in the assay. When this was done, however, it emerged that the neopterin determination in fresh sera, which are as a rule employed as samples, always gave measured values which were too high. For example, at a concentration of 5 nmol/l, the measured values were falsely more than 100% too high.
Elimination of interference from serum and/or other body fluids components can, as a rule, be achieved by using high dilutions of the serum, which however, is not possible because of the sensitivity required in this case. Furthermore, any possible denaturation of the sample by heat or the addition of denaturing agents is unsuitable for the neopterin determination. It turned out, however, that the interference declined during storage of the sera, and that the neopterin content can be measured correctly in older sera, as was ascertained from the very good correlations with other methods of determining neopterin. However, since the neopterin determination must as a rule be carried out in fresh sera, the falsely elevated values occurring under these circumstances are most disadvantageous for diagnostics.